The invention relates to optical memory devices using photochromic fluorescent protein moieties.
Photochromic materials can be used in a variety of applications, such as optical memory devices, described, for example, in Irie, M. & Mori, M. J. Org. Chem. 53:803 (1988); Parthenopoulos, D. A. & Rentzepis, P. M. Science 245:843 (1989); Hanazawa, M., et al. J. Chem. Soc. Chem. Commun. 206 (1992); Dvornikov, A. S., et al. J. Phys. Chem. 98:6746-6752 (1994); Dvornikov, A. S. & Rentzepis, P. M. Opt. Mem. Neur. Netw. 3:75-86 (1994); U.S. Pat. No. 4,471,470; and U.S. Pat. No. 5,325,324. The photochromic materials can be used in single-molecule optical storage at low temperatures. See, W. E. Moerner, Science 265:46 (1994). The photochromic materials have two states that are interconverted by irradiation. Optical devices based on bacteriorhodopsin (BR) are known, but do not allow for fluorescence detection.
The fluorescence behavior of wild-type (WT) green fluorescent protein (GFP) is known to have two absorption maxima, one at 395 nm, the other at 465 nm, but only one emission peak at 490 nm, indicating a common excited state. See, Heim, R., et al. Proc. Nat. Acad. Sci., USA 91:12501 (1994). The absorption peaks have been attributed to the neutral(state N) and anionic fluorophore states (state A.sup.-), respectively. The states can be interconverted by proton transfer between the fluorophore and Glu222. See, for example, Cubitt, A. B., et al. Trends in Biochem. Sci. 20:448 (1995); Chattoraj, M., et al. Proc. Nat. Acad. Sci., USA 93, 8362 (1996); and Brejc, K., et al. Proc. Nat. Acad. Sci., USA 94:2306-2311 (1997). Ser65 and Thr203 are particularly close to the chromophore in GFPs. See, Ormo, M., et al. Science 273:1392 (1996); Yang, F., et al. Nature Biotech. 14:1246 (1996). Consequently, these residues can strongly influence the photophysical properties of the protein. Alteration of Ser65 strongly favors ionization of the chromophore by hindering salvation and ionization of Glu222, whereas mutational loss of the Thr203 hydroxyl exerts a weaker opposing effect by reducing the salvation of the anionic form. See, Ormo, M., et al. Science 273:1392 (1996); Yang, F., et al. Nature Biotech. 14:1246 (1996). Aromatic residues at position 203 increase the peak excitation wavelength by 13-24 nm, probably by increasing the polarizability around the chromophore through p-p interactions. See, Ormo, M., et al. Science 273:1392 (1996).
In addition, new fluorescent proteins based on GFP have been identified by random screening of GFPs on plates. See, for example, Heim, R., et al. Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994); Ehrig, et al. FEBS Lett. 367:163-166 (1995); and Delagrave, et al. Bio/Technology 13:151-154 (1995). In each case, the bacteria were transformed with GFP cDNA (containing a large number of different mutations) and spread onto agar plates following standard molecular biological methods such as described in chapter 1 of Molecular Cloning, a Laboratory Manual, 2nd ed., by J Sambrook, E. F. Fritsch & T. Maniatis, Cold Spring Harbor Laboratory Press (1989). The resulting bacterial colonies were illuminated first with one excitation wavelength (i.e., 390-395 nm) then another excitation wavelength (i.e., 470-475 nm). Colonies that showed any unusual emission color or difference in brightness between the two excitation wavelengths were picked up by a sterile wire loop and grown further. The procedure can be carried out either by the eye or by a digital imaging system on a computer. See, for example, Youvan, D. C., et al. Methods in Enzymology, 246:732-748 (1995).